Electrochemical coagulation assay and device

ABSTRACT

Methods and devices for electrochemically detecting a change in the viscosity of a fluid are provided. In the subject methods, a fluid sample is introduced into an electrochemical cell having oppositely spaced apart working and reference electrodes. An electric potential is applied to the cell to first achieve a steady state cell current. A decrease in the steady state cell current is then detected and related to a change in viscosity of the sample. In many embodiments, the sample is blood and the change in viscosity is related to the onset of coagulation in the blood sample, and often the PT of the blood sample. Also provided are test strips, kits thereof and meters for use in practicing the subject methods.

FIELD OF THE INVENTION

The field of this invention is coagulation, and particularly coagulationtesting.

BACKGROUND

Coagulation is defined as a transformation of a liquid or sol into asoft, semi-solid or solid mass. Blood naturally coagulates to form abarrier when trauma or pathologic conditions cause vessel damage. Thereare two well-recognized coagulation pathways: the extrinsic orthromboplastin-controlled and the intrinsic orprothrombin/fibrinogen-controlled coagulation pathway. Both theextrinsic and intrinsic pathways result in the production of thrombin, aproteolytic enzyme which catalyzes the conversion of fibrinogen tofibrin.

Coagulation tests which measure a blood sample's ability to form a clotor coagulate have been developed and used to measure the ProthrombinTime (PT) of a blood sample. Such tests are commonly referred to as PTtests. PT tests find use in a number of different applications. Forexample, PT tests find use in monitoring patients undergoinganticoagulant therapy. Other situations where PT tests find use includetests to determine: acquired platelet function defect; congenitalplatelet function defects; congenital protein C or S deficiency; deepintracerebral hemorrhage; DIC (Disseminated intravascular coagulation);factor II deficiency; factor V deficiency; factor VII deficiency; factorX deficiency; hemolytic-uremic syndrome (HUS); hemophilia A; hemophiliaB; hemorrhagic stroke; hepatic encephalopathy; hepatorenal syndrome;hypertensive intracerebral hemorrhage; idiopathic thrombocytopenicpurpura (ITP); intracerebral hemorrhage; lobar intracerebral hemorrhage;placenta abruption; transient ischemic attack (TIA); Wilson's disease;and the like. As such, PT tests find use in a variety of differentapplications.

A number of different PT determination tests and devices have beendeveloped. Such devices and test protocols include both optical baseddevices, such as those described in U.S. Pat. No. 6,084,660; to R.Shartle; and electrochemical based devices, such as those described inU.S. Pat. Nos. 6,046,051; 6,060,323 and 6,066,504; all to A. Jina. Inthis latter group of patents a device is disclosed which is suitable forelectrochemical determination of a change of fluid viscosity in asample, where the device is characterized by the presence ofside-by-side electrodes. This configuration requires the use ofrelatively large volumes of sample and a measurement protocol thatimplements a time dependent deconvolution of the background response;i.e., signal is measured over time and is then distinguished overbackground. Thus, the protocols employed with Jina's devices are morecomplicated and perhaps less robust than the protocols used in thepresent invention described below.

While a number of different PT determination tests and devices have beendeveloped, there continues to be a need for additional protocols anddevices. Of particular interest would be the development of PT systemthat provided for rapid and accurate PT determinations with small samplevolumes using inexpensive device components, such as disposable reagentstrips. Of even greater interest would be the development of anelectrochemical device and protocol which exhibits the above desirableparameters, is suitable for use with small sample volumes and canprovide a simple-to-interpret signal that converges to a steady-statevalue.

RELEVANT LITERATURE

United States Patent of interest include: U.S. Pat. Nos. 6,084,660;6,066,504; 6,060,323, 6,046,051; 5,942,102; 5,916,522; 5,628,961;5,554,531; and 5,300,779. Also of interest are WO 97/18465; WO 95/06868;EP 974840 and GB 1 299 363.

SUMMARY OF THE INVENTION

Methods and devices for electrochemically detecting a change in theviscosity of a fluid are provided. In the subject methods, a fluidsample is introduced into an electrochemical cell having oppositelyspaced apart working and reference electrodes. An electric potential isapplied to the cell to first achieve a steady state cell current. Adecrease in the steady state cell current is then detected and relatedto a change in viscosity of the sample. In many embodiments, the sampleis blood and the change in viscosity is related to the onset ofcoagulation in the blood sample, and often the PT of the blood sample.Also provided are test strips, kits thereof and meters for use inpracticing the subject methods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides an exploded view of a reagent test strip according tothe subject invention.

FIG. 2 shows the time-current plot of a typical data set where blood isintroduced into a strip and the current is monitored with time.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and devices for electrochemically detecting a change in theviscosity of a fluid are provided. In the subject methods, a fluidsample is introduced into an electrochemical cell having oppositelyspaced apart working and reference electrodes. An electric potential isapplied to the cell to first achieve a steady state cell current. Adecrease in the steady state cell current is then detected and relatedto a change in viscosity of the sample. In many embodiments, the sampleis blood and the change in viscosity is related to the onset ofcoagulation in the blood sample, and often the PT of the blood sample.Also provided are test strips, kits thereof and meters for use inpracticing the subject methods.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, singular referencesinclude the plural, unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs.

Methods

As summarized above, the subject invention provides a method fordetermining a change in viscosity of a fluid sample. Often the subjectmethods provide a means for determining or detecting an increase in theviscosity of a fluid sample. The subject methods are sufficientlysensitive to detect an increase in viscosity that is less than about 1cps, and often less than about 0.5 cps in magnitude. As such, thesubject methods are sensitive methods for detecting a change inviscosity of a fluid sample.

Another feature of the subject methods is that they are electrochemicalmethods for determining a change, and often an increase, in theviscosity of a fluid sample. By electrochemical methods is meant thatthe subject methods employ a working and a reference electrode.Specifically, the subject methods employ a current produced between aworking and reference electrode and changes therein to determine achange in viscosity of the fluid sample, as described in greater detailbelow.

The first step in the subject methods is to introduce a quantity of thefluid to be assayed, i.e., a fluid sample, into an electrochemical cellthat includes oppositely spaced apart working and reference electrodes.The nature of the fluid may vary, so long as the fluid is a conductor,e.g., an electrolyte. In many embodiments, the fluid is an aqueousfluid, where of particular interest are physiological samples. Where thefluid is a physiological sample, in many embodiments the fluid is wholeblood, or a derivative thereof from which the coagulation/clotting time,and therefore PT time, can be derived.

The amount of fluid, e.g., blood, that is introduced into theelectrochemical cell varies, but is generally a small volume. As such,the volume of fluid introduced into the electrochemical cell typicallyranges from about 0.1 to 10 μL, usually from about 0.2 to 5.0 μL, andmore usually from about 0.3 to 1.6 μL. The sample is introduced into theelectrochemical cell using any convenient protocol, where the sample maybe injected into the electrochemical cell, allowed to wick into theelectrochemical cell, and the like, as may be convenient and dependingon the nature of the device/system in which the subject method ispracticed.

While the subject methods may be used, in principle, with any type ofelectrochemical cell having oppositely spaced apart working andreference electrodes, in many embodiments the subject methods employ anelectrochemical test strip. The electrochemical test strips employed inthese embodiments of the subject invention are made up of two opposingmetal electrodes separated by a thin spacer layer, where thesecomponents define a reaction area or zone that makes up theelectrochemical cell.

In certain embodiments of these electrochemical test strips, the workingand reference electrodes are generally configured in the form ofelongated rectangular strips. Typically, the length of the electrodesranges from about 1.9 to 4.5 cm, usually from about 2.0 to 2.8 cm. Thewidth of the electrodes ranges from about 0.07 to 0.76 cm, usually fromabout 0.24 to 0.60 cm. The working and reference electrodes typicallyhave a thickness ranging from about 10 to 100 nm and usually from about10 to 20 nm. FIG. 1 provides an exploded view of an electrochemical teststrip according to the subject invention.

The working and reference electrodes are further characterized in thatat least the surface of the electrodes that faces the reaction area ofthe electrochemical cell in the strip is a metal, where metals ofinterest include palladium, gold, platinum, silver, iridium, carbon(conductive carbon ink), doped tin oxide, stainless steel and the like.In many embodiments, the metal is gold or palladium. While in principlethe entire electrode may be made of the metal, each of the electrodes isgenerally made up of an inert support material on the surface of whichis present a thin layer of the metal component of the electrode. Inthese more common embodiments, the thickness of the inert backingmaterial typically ranges from about 25 to 500, usually 50 to 400 μm,e.g., from about 127 to 178 μm, while the thickness of the metal layertypically ranges from about 10 to 100 nm and usually from about 10 to 40nm, e.g. a sputtered metal layer. Any convenient inert backing materialmay be employed in the subject electrodes, where typically the materialis a rigid material that is capable of providing structural support tothe electrode and, in turn, the electrochemical test strip as a whole.Suitable materials that may be employed as the backing substrate includeplastics, e.g. PET, PETG, polyimide, polycarbonate, polystyrene,silicon, ceramic, glass, and the like.

A feature of the electrochemical test strips used in these embodimentsof the subject methods is that the working and reference electrodes asdescribed above face each other and are separated by only a shortdistance, such that the distance between the working and referenceelectrodes in the reaction zone or area of the electrochemical teststrip is extremely small. This minimal spacing of the working andreference electrodes in the subject test strips is a result of thepresence of a thin spacer layer positioned or sandwiched between theworking and reference electrodes. The thickness of this spacer layer mayrange from 50 to 750 μm and is often less than or equal to 500 μm, andusually ranges from about 100 to 175 μm, e.g., 102 to 153 μm. The spacerlayer is cut so as to provide a reaction zone or area with at least aninlet port into the reaction zone, and generally an outlet port out ofthe reaction zone as well. The spacer layer may have a circular reactionarea cut with side inlet and outlet vents or ports, or otherconfigurations, e.g. square, triangular, rectangular, irregular shapedreaction areas, etc. The spacer layer may be fabricated from anyconvenient material, where representative suitable materials includePET, PETG, polyimide, polycarbonate, and the like, where the surfaces ofthe spacer layer may be treated so as to be adhesive with respect totheir respective electrodes and thereby maintain the structure of theelectrochemical test strip. Of particular interest is the use of adie-cut double-sided adhesive strip as the spacer layer.

The electrochemical test strips used in these embodiments of the subjectinvention include a reaction zone or area that is defined by the workingelectrode, the reference electrode and the spacer layer, where theseelements are described above. Specifically, the working and referenceelectrodes define the top and bottom of the reaction area, while thespacer layer defines the walls of the reaction area. The volume of thereaction area typically ranges from about 0.1 to 10 μL, usually fromabout 0.2 to 5.0 μL, and more usually from about 0.3 to 1.6 μL. Asmentioned above, the reaction area generally includes at least an inletport, and in many embodiments also includes an outlet port. Thecross-sectional area of the inlet and outlet ports may vary as long asit is sufficiently large to provide an effective entrance or exit offluid from the reaction area, but generally ranges from about 9×10⁻⁴ to5×10⁻³ cm², usually from about 1.3×10⁻³ to 2.5×10⁻³ cm².

In many embodiments, a reagent system is present in the reaction area,where the reagent system interacts with components in the fluid sampleduring the assay. For example, in embodiments where the subject methodsare used to detect a coagulation event, e.g., to measure PT of a sample,the reaction area or zone includes a reagent system that at leastincludes a redox couple, and often also includes a coagulationcatalyzing agent.

The redox couple of the reagent composition, when present, is made up ofone or more redox couple agents. A variety of different redox coupleagents are known in the art and include: ferricyanide, phenazineethosulphate, phenazine methosulfate, pheylenediamine,1-methoxy-phenazine methosulfate, 2,6-dimethyl-1,4-benzoquinone,2,5-dichloro-1,4-benzoquinone, ferrocene derivatives, osmium bipyridylcomplexes, ruthenium complexes, and the like. In many embodiments, redoxcouples of particular interest are ferricyanide, and the like.

In many embodiments, the reagent composition also includes a coagulationcatalyzing agent. By coagulation catalyzing agent is meant one or morecomponents or reactants that participate or interact with componentspresent in the fluid sample, e.g., whole blood, to initiate the clottingprocess in the blood sample. For PT assays, the coagulation catalyzingagent generally comprises thromboplastin, which thromboplastin may bepurified from a naturally occurring source, e.g., an aqueous extract ofacetone dried brain tissue, or synthetic recombinant thromboplastin(r-DNA thromboplastin), which generally includes purified recombinanttissue factor protein and a purified artificial lipid component. Arepresentative coagulation catalyzing agent is thromboplastin-XS withcalcium sold under the trade name INNOVIN® by Dade International, MiamiFla.

Other reagents that may be present in the reaction area includebuffering agents, e.g. citraconate, citrate, malic, maleic, phosphate,“Good” buffers and the like. Yet other agents that may be presentinclude: divalent cations such as calcium chloride, and magnesiumchloride; surfactants such as Triton, Macol, Tetronic, Silwet, Zonyl,and Pluronic; stabilizing agents such as albumin, sucrose, trehalose,mannitol, and lactose.

The reagent system, when present, is generally present in dry form. Theamounts of the various components may vary, where the amount of theoxidized redox couple component typically ranges from about 5 to 1000mM, usually from about 90 to 900 mM; the reduced redox couple componenttypically ranges from about 1 to 20 mM, usually from about 5 to 15 mM;the amount of buffer typically ranges from about 0 to 300 mM, usuallyfrom about 50 to 100 mM; and the amount of coagulation catalyzing agentcomponent typically ranges from about 0.005 to 50 mg/cm², usually fromabout 0.05 to 5 mg/cm² . The overall mass of dry reagent present in thereaction area or zone in these embodiments generally ranges from about 4to 700 ng/cm², usually from about 8 to 350 ng/cm².

A representative test strip for use in the subject methods is depictedin exploded view in FIG. 1.

Following sample introduction into the electrochemical cell, a constantelectric potential is applied to the cell in a manner sufficient toproduce a steady state current between the working and referenceelectrodes of the cell. More specifically, a constant electric potentialis applied between the working and the reference electrodes in a mannerthat produces a steady state current between the two electrodes. Themagnitude of the applied electric potential generally ranges from about0 to −0.6 V, usually from about −0.2 to −0.4 V. In many embodimentswhere the electrochemical cell includes a redox couple, as describedabove, application of the constant electrical potential as describedabove results in the production of a steady state current described bythe following, formula:

i _(ss) =n2FADCo/L;

where:

n is equal to the number of electrons transferred;

F is Faraday's constant, i.e., 9.6485×10⁴C/mol;

A is the area of the working electrode;

D is the diffusion coefficient of the cell, where this coefficient maybe determined from Fick's first law, i.e. J(x,t)=−D^(dCo(x,t))/_(dx)where j is flux, x is the position from the electrode, and t is time;

Co is the redox couple concentration, e.g., the ferrocyanideconcentration; and

L is distance between the electrodes, e.g., the spacer thickness.

The overall time period required to obtain the requisite steady statecurrent, as described above, is relatively short in certain embodiments.In such embodiments, the total amount of time required to obtain thesteady state current, i.e., the period from sample entry to the cell toestablishment of the steady state current, is less than about 15seconds, usually less than about 10 seconds; and often ranges from about4 to 15 seconds.

FIG. 2 shows the time-current plot of a typical data set where blood isintroduced into a strip and the current is monitored with time.

The next step in the subject methods is to detect a change in the steadystate current and relate this change to a change in viscosity of thesample. In many embodiments, the change that is detected is a decreasein the steady state current. The magnitude of the decrease in the steadystate current that is detected in this step is at least about 2%, andusually at least about 10%, where the magnitude of the decrease in manyembodiments ranges from about 2 to 90%. In other embodiments, ofinterest is the rate of change between two steady state values, onebefore and one after the coagulation event, and the relation of thischange in rate to the presence of the coagulation event.

The detection of the above described decrease in steady state current isthen related to an increase in viscosity of the fluid sample in theelectrochemical cell. Relatively small increases in viscosity result ina detectable decrease in the steady state current and thus can bedetected by the subject methods, where the magnitude of the increase inviscosity may be as small as 0.5 cps or smaller in certain embodiments.

In many embodiments where the sample present in the electrochemical cellis whole blood and the reagent composition includes a coagulationcatalyzing agent, the increase in viscosity is then related to the onsetof coagulation in the blood sample, i.e., the occurrence of acoagulation event or blood clotting in the blood sample. In certainembodiments, the increase in viscosity and concomitant detection of theonset of coagulation in the blood sample being assayed is employed todetermine the PT of the blood sample. In these embodiments, the periodextending from the initial sample introduction into the reaction area orzone and/or the establishment of a steady state current and increase inviscosity/onset of coagulation is determined and the PT of the bloodsample is derived from this time period. The time at which sample entersthe electrochemical cell may be detected using any convenient protocol,where particular protocols employed may depend, at least in part, on thenature of the meter device employed with the electrochemical cell. Incertain embodiments, the time that sample is introduced directly intothe reaction cell can be manually recorded. Alternatively, the meter mayautomatically detect sample introduction into the electrochemical cell,e.g., by detecting an initial decrease in the voltage required toachieve a constant current between the working and reference electrodesof the cell. (See U.S. application Ser. No. 9/333793, filed Jun. 15,1999, incorporated herein by reference.) Other protocols for sampledetection in the cell may also be employed.

The above computational steps of the subject method, e.g., relation ofthe time period from sample introduction to onset of coagulation to thePT of the blood sample, may be accomplished manually or through the useof an automated computing means, where in many embodiments the use of anautomated computing means, such as is described in connection with thesubject devices discussed below, is of interest.

The above described protocol may be carried out at room temperature orat an elevated temperature. Typically, the above protocol is carried outat a temperature ranging from about 20 to 40° C., e.g., about 37° C.

The above described methods find use in any application where thedetermination of a viscosity change in a fluid sample is desirable. Assuch, the subject methods suited for use in the determination of PT of ablood sample, and as such find use in any application where thedetermination of PT is desired, e.g., those applications described inthe Background Section, supra.

Devices

Also provided by the subject invention are meters for use in practicingthe subject invention. The subject meters are typically meters formeasuring a change in viscosity of fluid sample, and are meters formeasuring the PT of a blood sample in many embodiments. The subjectmeters typically include: (a) a means for applying an electric potentialto an electrochemical cell into which the sample has been introduced;(b) a means for measuring cell current in the cell, including a steadystate current in the cell; (c) a means for detecting a change in thesteady state current in the cell, e.g., a decrease in the steady statecurrent of the cell; and (d) a means for relating the change in steadystate current to a change in viscosity of the cell, e.g., a means forrelating a decrease in steady state current in the cell to an increasein viscosity of fluid in the cell.

The means for applying.an electric potential to the electrochemicalcell, means for measuring a steady state current in the cell and meansfor detecting a change in the steady state current in the cell may beany convenient means, where representative means are described in WO97/18465 and U.S. Pat. No. 5,942,102; the disclosures of which areherein incorporated by reference. See also U.S. Pat. Nos. 6,066,504;6,060,323; 6,046,051; the disclosures of which are herein incorporatedby reference. The means for relating the change in steady state currentto a change in viscosity is typically a computing means present in themeter which is capable of relating the measured change in steady statecurrent to a change in viscosity of the fluid sample. In manyembodiments, this means is further a means for relating the change incurrent/viscosity to the onset of coagulation, and is often a means fordetermining the PT of a blood sample. See e.g., U.S. Pat. No. 6,066,504;the disclosure of which is herein incorporated by reference.

Kits

Also provided are kits for use in practicing the subject methods. Thekits of the subject invention at least include an electrochemicalreagent test strip, as described above. The subject kits may furtherinclude a means for obtaining a physiological sample. For example, wherethe physiological sample is blood, the subject kits may further includea means for obtaining a blood sample, such as a lance for sticking afinger, a lance actuation means, and the like. In addition, the subjectkits may include a calibration means for calibrating the instrument,e.g., a control solution or standard, e.g., a coagulation controlsolution that has a known PT time. In certain embodiments, the kits alsoinclude an automated instrument, as described above, for detecting theamount of product produced on the strip following sample application andrelating the detected product to the amount of analyte in the sample.Finally, the kits include instructions for using the subject kitcomponents in the determination of an analyte concentration in aphysiological sample. These instructions may be present on one or moreof the packaging, a label insert, containers present in the kits, andthe like.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

I. Electrochemical Test Strip Preparation

An electrochemical test strip consisting of two metallized electrodesoriented in a sandwich configuration was prepared as follows. The toplayer of the test strip was a gold sputtered Mylar strip. The middlelayer was a double-sided adhesive with a punched hole that defined thereaction zone or area. The punched hole was a circle with two juxtaposedrectangular inlet and outlet channels. The bottom layer of the teststrip was sputtered palladium on Mylar. A reagent of citraconate buffer,ferricyanide, ferrocyanide and relipidated recombinant tissue factor wasink jetted on the palladium sputtered surface. The amount of reagent inkjetted onto the palladium sputtered surface was 597 ng/cm². As such, theamount of citraconate buffer was 120 ng/cm² ferricyanide was 460 ng/cm²,the amount of ferrocyanide was 8 ng/cm² and the amount of recombinanttissue factor was 9 ng/cm². An exploded view of the test strip is shownin FIG. 1.

II. Detection of PT

The above described strip is employed to determine the PT of a bloodsample as follows. A 1.5 μl blood sample is introduced into the reactionarea or zone of the test strip and the sample introduction time isrecorded. A constant potential of −0.3 V is applied between the workingand reference electrodes, and the resultant current between the twoelectrodes is monitored. The appearance of a steady state current isfirst detected, followed by a decrease in the steady state current. Thetime period from the initial sample introduction to the decrease insteady state current is determined and then related to the PT of theblood sample. FIG. 2 shows the time-current plot of the data set whereblood is introduced into a strip and the current is monitored with time.

The above results and discussion demonstrate that subject inventionprovides a simple and powerful tool to determine the PT of a bloodsample. Advantages of the subject methods over non-electrochemical basedcoagulation detection methods include use of low cost materials and theopportunity to use wide variety of controls, including plasma basedcontrols. Additional advantages of the subject invention include theability to employ small sample volumes and the fact that theelectrochemical measurements made by the subject methods provide asimple-to-interpret signal that converges to a steady-state value. Yetanother advantage is the ability to use low cost electrochemical basedmeters, which provide for significant cost savings. As such, the subjectinvention represents a significant contribution to the art.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. The citation of any publication is for its disclosure priorto the filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A method for detecting a change in the viscosityof a fluid sample, said method comprising: (a) introducing a sample intoan electrochemical cell comprising a redox couple and oppositely spacedapart working and reference electrodes; (b) applying an electricpotential to said reaction cell to produce a peak in current, followedby a steady state current between said oppositely spaced apartelectrodes; (c) detecting a change from said steady state current; and(d) relating said change in steady state current to a change inviscosity of said fluid sample.
 2. The method according to claim 1,wherein said change from steady state current is a decrease.
 3. Themethod according to claim 1, wherein said change in viscosity is anincrease.
 4. The method according to claim 1, wherein said fluid sampleis a physiological sample.
 5. The method according to claim 4, whereinsaid physiological sample is blood.
 6. The method according to claim 5,wherein said method further comprises relating said change in viscosityto the prothrombin time (PT) of said blood.
 7. A method for detectingthe onset of coagulation of a blood sample, said method comprising: (a)introducing said blood sample into an electrochemical cell comprising:(i) oppositely spaced apart working and reference electrodes; and (ii) areagent mixture comprising a redox couple; (b) applying an electricpotential to said reaction cell to produce a peak in current, followedby a steady state current between said oppositely spaced apartelectrodes; (c) detecting a change from said steady state current; and(d) relating said change in steady state current to the onset ofcoagulation in said blood sample.
 8. The method according to claim 7,wherein said change is a decrease.
 9. The method according to claim 7,wherein said reagent comprises a coagulation catalyzing agent.
 10. Themethod according to claim 9, wherein said coagulation catalyzing agentcomprises thromboplastin.
 11. The method according to claim 9, whereinsaid method further comprises relating said onset of coagulation to theprothrombin time of said blood sample.